Members of the protein tyrosine phosphatase (PTP) superfamily, all contain the highly conserved active site motif, Cys-x5- Arg (Cx5R), and are key mediators of a variety of cellular processes including growth, differentiation, motility, metabolism and programmed cell death. This proposal focuses on two novel PTPs whose functions are potentially linked to energy metabolism in the cell. The first two specific aims are directed at understanding laforin, a phosphatase mutated in the most severe form of progressive myoclonus epilepsy, Lafora's disease (LD). The hallmark of LD is the accumulation of starch-like polyglucosans called Lafora bodies. My laboratory has recently shown that malin, an E3 ligase also mutated in a subset of LD patients, interacts with and ubiquitinates laforin. Specific Aim 1 outlines our intentions to fully characterize laforin's phosphatase activity. We have optimized bacterial expression and purification of laforin for use in these kinetic analyses as well as for crystallization efforts to determine the first structure of laforin. Specific Aim 2 focuses on the modification of laforin by malin and the cellular consequences of this modification. Successful completion of these specific aims will give us a better understanding of the etiology of LD. The remaining three specific aims are designed to characterize FLIP, the first known mitochondrial PTP. Given the absence of precedent for PTPs in this organelle, and a nearly complete lack of known mitochondrial signaling pathways involving reversible phosphorylation, we will first perform a thorough characterization of PLIP in Specific Aim 3. This aim includes pinpointing PLIP to a specific submitochondrial compartment, establishing its mechanism of import, and determining a tissue expression profile. Specific Aim 4 is an effort to elucidate the mitochondrial function(s) of PLIP using RNAi strategies in tissue culture cells and conditional knock-out strategies in mice. PLIP's potential function in cell culture will be evaluated with respect to glucose stimulated insulin secretion (GSIS), apoptosis, and the general - bioenergetic status of mitochondria. Our Cre-lox knockout mouse strategy will afford us the opportunity of ablating PLIP expression in a variety of different tissues enabling us to evaluate our cell culture data in the context of the whole animal. Specific Aim 5 is designed to identify the cellular substrate(s) for PLIP, in part through leveraging our powerful RNAi technique in Drosophila S2 cells. Successful completion of Specific Aims 3-5 will give us a better understanding of PLIP's role in mitochondrial function and help to reveal mitochondria as important centers for cell signaling.